|Glycoproteins were fractionated from serum using the different lectin spin columns in glycoprotein fractionation kits and analyzed by SDS-PAGE followed by silver staining. [A] Elution steps 1–3 and 4–6 from Total Lectin Spin Columns in the Total Glycoprotein Kit. [B] Eluted glycoproteins from ConA, GNA, and LCH Spin Columns in the Mannose Glycoprotein Kit. [C] Eluted glycoproteins from WGA, SNA, and MAL Spin Columns in the Sialic Glycoprotein Kit. [D] Eluted glycoproteins from AIL and PNA Spin Columns in the O-Glycan Glycoprotein Kit.|

Glycosylated proteins in the sample bind to lectins immobilized on the resin in the spin columns. After washing, proteins are eluted in two stages by sequential addition of buffers containing sugars which compete with bound proteins for the lectin binding sites.

Procedure

The sample is diluted and applied to the spin column (see figure "Glycoprotein fractionation procedure"). After a short incubation, non-glycosylated proteins are removed by centrifugation. Glycosylated proteins are eluted by application of elution buffer and centrifugation.

Applications

Glycosylation plays a vital role in a wide range of cellular processes such as cell adhesion and signaling, stabilization of protein structure and function, protein trafficking and sorting, and oncogenesis. Several diseases (e.g., rheumatoid arthritis, muscular dystrophy) may be caused by a defect in protein glycosylation. The Qproteome Glycoprotein Fractionation Kit offers highly specific separation of glycoproteins according to the structure of their glycan moieties, and permits profiling of glycoproteins in cells grown under different conditions. Purified glycoproteins are suitable for direct analysis using blotting procedures, 2D-PAGE, or mass spectrometry methods.